Compositions and methods of use for therapeutic antibodies

ABSTRACT

The present invention relates to antibodies that specifically bind to the BAFF receptor (BAFFR). The invention more specifically relates to specific antibodies that are BAFFR antagonists with in vivo B cell depleting activity and compositions and methods of use for said antibodies to treat pathological disorders that can be treated by killing or depleting B cells, such as systemic lupus erythematosus or rheumatoid arthritis or other autoimmune diseases or lymphomas, leukemias and myelomas.

This application is a continuation of U.S. patent application Ser. No.12/503,175, filed Jul. 15, 2009 which claims benefit under 35 U.S.C.§119(a)-(d) or (f) or 365(b) of EP Application No. 08160671.7, filedJul. 17, 2008, and EP Application No. 09160326.6, filed May 15, 2009,the contents of which are incorporated herein by reference in theirentirety.

The present invention relates to antibodies that specifically bind tothe BAFF receptor (BAFFR). The invention more specifically relates tospecific antibodies that are BAFFR antagonists with in vivo B celldepleting activity and compositions and methods of use for saidantibodies to treat pathological disorders that can be treated bykilling or depleting B cells, such as systemic lupus erythematosus orrheumatoid arthritis or other autoimmune diseases or lymphomas,leukemias and myelomas.

BAFFR (also known as BR3, TNFRSF13C, or CD268) is a member of the tumornecrosis factor receptor superfamily. It is expressed predominantly onB-lymphocytes and on a subset of T-cells. BAFFR specifically binds thetumor necrosis factor family member BLyS (also known as BAFF, CD257,TALL-1, THANK, TNFSF13B, ZTNF4) which can be expressed by a variety ofdifferent cell types, most notably myeloid cells. Functionally, theBLyS/BAFFR ligand-receptor pair is critically involved in the maturationof immature transitional B-cells and for survival, migration andactivation of mature B-cells including isotype class switching. BLyS canact alone or in concert with B-cell receptor (BCR), interleukin-4,interleukin-21 or CD40 ligand. Due to the presence of BAFFR on someT-cells, BLyS can act as co-stimulatory factor for T-cell activation.BLyS can also bind to two additional receptors found on B-cells, TACIand BCMA.

Overexpression of BLyS or BAFFR in mice leads to B-cell hyperplasia anddevelopment of systemic auto-immunity with classical features ofsystemic lupus erythematosus (SLE). In addition, diseased (NZBxNZVV)F1and autoimmune MRL-Ipr/lpr mice which represent animal models of SLEcontain increased BLyS concentrations in the serum and BLyS levelscorrelate with disease progression. Increased levels of BLyS are alsofound in human patients suffering from SLE, rheumatoid arthritis,Sjögren's syndrome, Wegener's granulomatosis and B-cell malignancies.Furthermore, the disease phenotype in animal models of auto-immunediseases such as rheumatoid arthritis (e.g. collagen induced arthritis),SLE and multiple sclerosis (e.g. experimental auto-immuneencephalomyelitis) can be partially reverted by BLyS blockade withsoluble receptor fusion proteins. Similarly, treatment with BAFFR:Fcfusion protein inhibits chronic graft-versus-host disease (cGVHD) byblocking B-cell survival. Clinical efficacy data with a blockinganti-BLyS antibody in rheumatoid arthritis and SLE patients underscorethe pathogenic role of BLyS in these auto-immune disorders.

BLyS induced signaling also appears to be involved in survival ofmalignant B-cells. Apoptosis of B-CLL cells can be rescued by additionof recombinant BLyS or APRIL. Conversely, apoptosis of B-CLL cells isaugmented by adding soluble BAFFR fusion proteins or by anti-APRILantibodies, indicating that BLyS and APRIL could serve as autocrinegrowth factors for malignant B-cells. BAFFR is expressed on a variety ofdiseased tissue including multiple myeloma and non-Hodgkin's lymphoma.Currently available treatments for these autoimmune diseases areimmunosuppressants with severe side effects that do not cure the diseasebut aim at improving the signs and symptoms of the disease(disease-modifying drugs). Most of the immunosuppressants currently usedin SLE and RA like corticosteroids, cyclophosphamide, methotrexate andazathioprin lead to a general anti-inflammatory effect that carries therisk of severe infections since it affects all effector arms of theimmune system. Therefore, there is still a need for compositions andmethods to treat SLE and/or RA and other related autoimmune diseases,such as agents that interfere with BAFFR signaling in which BLyS issuspected to contribute to disease.

Therefore, in one aspect, the invention provides an antibody or afunctional protein comprising an antigen-binding portion of saidantibody for a target in BAFFR polypeptide (SEQ ID NO:87), characterizedin that the antibody or functional protein specifically binds to BAFFRpolypeptide. In one embodiment, the antibody or functional protein isfrom a mammal, having an origin such as human or camelid, or is ahumanized antibody. In a particular embodiment, the anti-BAFFR antibodyis characterized as having antigen-binding region that is specific forthe target protein BAFFR and binds to BAFFR or a fragment of BAFFR.

In one embodiment, the antibodies according to the invention are BAFFRantagonists with no or low agonistic activity. In certain embodiments,the antibody or functional fragment binds the target protein BAFFR anddecreases or inhibits BLyS binding to BAFFR. In a related embodiment,the antibody or functional fragment inhibits BLyS induced human B cellproliferation, and/or IgG1 production.

In another embodiment, the antibodies according to the invention depleteB cell in vitro and in vivo. More preferably, the antibodies of theinvention are BAFFR antagonists with no agonistic activity and depletehuman B cell in vitro and in vivo.

The binding may be determined by one or more assays that can be used tomeasure an activity which is either antagonism or agonism by theantibody. Preferably, the assays measure at least one of the effects ofthe antibody on BAFFR that include: BLyS induced human B cellproliferation, IgG1 production and/or human B cell depleting activity.

In another embodiment, the invention provides antibodies thatspecifically bind to BLyS binding region of BAFFR. In a relatedembodiment, the invention provides antibodies that bind to a region ofBAFFR between amino acids 17 and 43 of SEQ ID NO:87 and for example, itbinds at least to PTPCVPAECFDLLVRHCVACGLLR (SEQ ID NO 88).

According to another particular embodiment, the antibodies bind to BAFFRwith a K_(D) of 100 nM or less, 10 nM or less, 1 nM or less, inhibitBLyS induced human B cell proliferation with an IC₅₀ around 10 nM orless, 1 nM or less or 100 pM or less and deplete B cells in vitro withan EC₅₀ of 10 nM or less, 1 nM or less or 100 pM or less.

In another related embodiment, the antibodies reduce the percentage of Bcells in blood and tissue in vivo up to 70%, preferably 80%, and morepreferably 90% in a mouse model as compared to untreated controlanimals.

In some particular embodiments, the antibodies of the invention do notcross-react with a BAFFR related protein, and more particularly do notcross-react with human TACI or BCMA receptor.

In another related embodiment, the antibodies according to the inventionare fully human or humanized IgG1 antibodies with antibody dependentcellular cytotoxicity (ADCC) activity and bind to a region of BAFFRcomprised between amino acids 17 and 43 of SEQ ID NO:87, and forexample, at least the following peptides PTPCVPAECFDLLVRHCVACGLLR (SEQID NO 88) and deplete B cells in vitro with an EC₅₀ of 10 nM or less, 1nM or less or 100 pM or less.

In another related embodiment, the antibodies according to the inventionare human antibodies produced by recombinant expression in a cell linelacking fucosyltransferase, for example a mammalian cell line withdeficient expression of the FUT8 gene encoding fucosyltransferase,thereby increasing ADCC activity as compared with wild type cellsexpressing the FUT8 gene.

The present invention relates to isolated antibodies, particularly humanor humanized antibodies, that interfere with, decrease or inhibit BLySbinding to BAFFR and that depletes B cells in vitro and in vivo. Incertain embodiments, the antibodies of the invention are derived fromparticular heavy and light chain sequences and/or comprise particularstructural features such as CDR regions comprising particular amino acidsequences. The invention provides isolated antibodies, methods of makingsuch antibodies, immunoconjugates and multivalent or multispecificmolecules comprising such antibodies and pharmaceutical compositionscontaining the antibodies, immunoconjugates or bispecific molecules ofthe invention. The invention also relates to methods of using theantibodies to inhibit, e.g., antagonize, function of BAFFR in order todelay, prevent, prevent the onset of, or inhibit development of adisorder or condition associated with the presence of BLyS and/or BAFFR,for example, resulting in the treatment of a pathological disorder thatis mediated by BAFFR or that can be treated by killing or depleting Bcells; for example, an autoimmune disease such as systemic lupuserythematosus (SLE) or rheumatoid arthritis (RA) or B cell neoplasm suchas lymphomas, leukemias or myelomas. Thus, such antibodies, antibodyfragments or antigen-binding proteins may find use prophylactically,preventatively, or as part of a treatment method.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” or “signaling activity” refers to abiochemical causal relationship generally initiated by a protein-proteininteraction such as binding of a growth factor to a receptor, resultingin transmission of a signal from one portion of a cell to anotherportion of a cell. In general, the transmission involves specificphosphorylation of one or more tyrosine, serine, or threonine residueson one or more proteins in the series of reactions causing signaltransduction. Penultimate processes typically include nuclear events,resulting in a change in gene expression.

The term BAFFR or BAFF receptor refers to human BAFFR as defined in SEQID NO: 87. PCT Patent Publications WO200004032 and WO2006073941 refer toanti-BAFFR antibodies in general. WO2006073941 describes specificanti-BAFFR antibodies.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. A naturally occurring “antibody” is a glycoproteincomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as V_(L)) and a lightchain constant region. The light chain constant region is comprised ofone domain, C_(L). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each V_(H) and V_(L) is composed of three CDRsand four FRs arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., a portion of BAFFR). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding region” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds BAFFRis substantially free of antibodies that specifically bind antigensother than BAFFR). An isolated antibody that specifically binds BAFFRmay, however, have cross-reactivity to other antigens, such as BAFFRmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis, forexample, as described in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The structures and locations of immunoglobulin variable domains, e.g.,CDRs, may be defined using well known numbering schemes, e.g., the Kabatnumbering scheme, the Chothia numbering scheme, a combination of Kabatand Chothia (AbM), etc. (see, e.g., Sequences of Proteins ofImmunological Interest, U.S. Department of Health and Human Services(1991), eds. Kabat et al.; Al Lazikani et al. (1997) J. Mol. Bio.273:927 948).

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG2) that is provided by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody that “specifically binds to BAFFRpolypeptide” is intended to refer to an antibody that binds to humanBAFFR polypeptide with a K_(D) of 100 nM or less, 10 nM or less, 1 nM orless. An antibody that “cross-reacts with an antigen other than BAFFR”is intended to refer to an antibody that binds that antigen with a K_(D)of 0.5×10⁻⁸ M or less, 5×10⁻⁹ M or less, or 2×10⁻⁹ M or less. Anantibody that “does not cross-react with a particular antigen” isintended to refer to an antibody that binds to that antigen, with aK_(D) of 1.5×10⁻⁸ M or greater, or a K_(D) of 5-10×10⁻⁸ M or 1×10⁻⁷ M orgreater. In certain embodiments, such antibodies that do not cross-reactwith the antigen exhibit essentially undetectable binding against theseproteins in standard binding assays.

As used herein, the term “antagonist antibody” is intended to refer toan antibody that reduces, decreases and/or inhibits BAFFR inducedsignaling activity in the presence of BLyS in a human B cell assay suchas human B cell proliferation assay or human B cell IgG1 productionassay. Examples of human B cell proliferation assay and IgG1 productionassay are described in more details in the examples below. In someembodiments, the antibodies reduce, decrease or inhibit BLyS inducedactivity as measured in a human B cell proliferation assay at an IC₅₀ of10 nM or less, 1 nM or less, or 100 pM or less. In some embodiments, theantibodies inhibit BLyS induced activity as measured in a IgG1production assay at an IC₅₀ of 10 nM or less, 1 nM or less, or 100 pM orless.

As used herein, an antibody with “no agonistic activity” is intended torefer to an antibody that does not significantly increase BAFFR mediatedsignaling activity in the absence of BLyS in a cell-based assay, such ashuman B cell proliferation assay. Such assays are described in moredetails in the examples below.

As used herein, an antibody that depletes B cells in vitro is intendedto refer to an antibody that depletes B cells with an EC₅₀ of 10 nM orless, preferably with an EC₅₀ of 1 nM or less, more preferably with anEC₅₀ of 100 pM, or less, as measured in a human B cell depletion assay(ADCC). Such assays are described in more details in the examples below.

As used herein, an antibody that depletes B cells in vivo is intended torefer to an antibody that reduces in vivo the percentage of B cells upto 70%, preferably 80% and more preferably 90%, as measured byfluorescence activated cell sorting (FACS) of B cells. Such assays aredescribed in more details in the examples below.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK. (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system.

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

As used herein, the term “ADCC” or “antibody dependent cellularcytotoxicity” activity refers to human B cell depleting activity. ADCCactivity can be measured by the human B cell depleting assays describedabove.

In order to obtain a higher avidity probe, a dimeric conjugate (twomolecules of an antibody protein coupled to a FACS marker) can beconstructed, thus making low affinity interactions (such as with thegermline antibody) more readily detected by FACS. In addition, anothermeans to increase the avidity of antigen binding involves generatingdimers, trimers or multimers of any of the constructs described hereinof the anti-BAFFR antibodies. Such multimers may be generated throughcovalent binding between individual modules, for example, by imitatingthe natural C-to-N-terminus binding or by imitating antibody dimers thatare held together through their constant regions. The bonds engineeredinto the Fc/Fc interface may be covalent or non-covalent. In addition,dimerizing or multimerizing partners other than Fc can be used in BAFFRhybrids to create such higher order structures. For example, it ispossible to use multimerizing domains such as trimerizing domaindescribed in Borean (WO2004039841) or pentamerizing domain described inpublished patent application WO98/18943.

As used herein, the term “selectivity” for an antibody refers to anantibody that binds to a certain target polypeptide but not to closelyrelated polypeptides.

As used herein, the term “high affinity” for an antibody refers to anantibody having a K_(D) of 1 nM or less for a target antigen. As usedherein, the term “subject” includes any human or nonhuman animal.

The term “nonhuman animal” includes all vertebrates, e.g., mammals andnon-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows,chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a cell of Trichoderma, a ChineseHamster Ovary cell (CHO) or a human cell. The optimized nucleotidesequence is engineered to retain completely or as much as possible theamino acid sequence originally encoded by the starting nucleotidesequence, which is also known as the “parental” sequence. The optimizedsequences herein have been engineered to have codons that are preferredin CHO mammalian cells; however optimized expression of these sequencesin other eukaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

Various aspects of the invention are described in further detail in thefollowing subsections.

Standard assays to evaluate the binding ability of the antibodies towardBAFFR of various species are known in the art, including for example,ELISAs, western blots and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis. Assays to evaluate the effects of theantibodies on functional properties of BAFFR (e.g., receptor binding,preventing or inducing human B cell proliferation or IgG production) aredescribed in further detail in the Examples.

Accordingly, an antibody that “inhibits” one or more of these BAFFRfunctional properties (e.g., biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(e.g., or when a control antibody of irrelevant specificity is present).An antibody that inhibits BAFFR activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of theinvention may inhibit greater than 95%, 98% or 99% of BAFFR functionalactivity.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to BAFFR in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to BAFFR, and therefore whether it can be said to cross-blockaccording to the invention, can be determined using standard competitionbinding assays. One suitable assay involves the use of the Biacoretechnology (e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala,Sweden)), which can measure the extent of interactions using surfaceplasmon resonance technology. Another assay for measuring cross-blockinguses an ELISA-based approach.

Further details on both methods are given in the Examples.

According to the invention, a cross-blocking antibody or other bindingagent according to the invention binds to BAFFR in the described BIAcorecross-blocking assay such that the recorded binding of the combination(mixture) of the antibodies or binding agents is between 80% and 0.1%(e.g. 80% to 4%) of the maximum theoretical binding, specificallybetween 75% and 0.1% (e.g. 75% to 4%) of the maximum theoreticalbinding, and more specifically between 70% and 0.1% (e.g. 70% to 4%),and more specifically between 65% and 0.1% (e.g. 65% to 4%) of maximumtheoretical binding (as defined above) of the two antibodies or bindingagents in combination

An antibody is defined as cross-blocking in the ELISA assay as describedin the Examples, if the solution phase anti-BAFFR antibody is able tocause a reduction of between 60% and 100%, specifically between 70% and100%, and more specifically between 80% and 100%, of the BAFFR detectionsignal (i.e. the amount of BAFFR bound by the coated antibody) ascompared to the BAFFR detection signal obtained in the absence of thesolution phase anti-BAFFR antibody (i.e. the positive control wells).

Recombinant Antibodies

Antibodies of the invention include the human recombinant antibodies,isolated and structurally characterized as described, in the Examples.The V_(H) amino acid sequences of isolated antibodies of the inventionare shown in SEQ ID NOs: 50-56. The V_(L) amino acid sequences ofisolated antibodies of the invention are shown in SEQ ID NOs: 43-49respectively. Examples of preferred full length light chain amino acidsequences of antibodies of the invention are shown in SEQ ID NO:71-74.Examples of preferred full length heavy chain amino acid sequences ofantibodies of the invention are shown in SEQ ID NO:75-78 respectively.Other examples of preferred full length heavy and light chain amino acidsequences of antibodies are those encoded by corresponding DNA sequencescontained in plasmids pBW510 and pBW512 as deposited by Novartis PharmaAG, Forum 1, CH-4002 Base. Switzerland, in DSMZ on Apr. 29, 2009 withaccession number DSM22542 and DSM22543 respectively. Other antibodies ofthe invention include amino acids that have been mutated by amino aciddeletion, insertion or substitution, yet have at least 60, 70, 80, 90 or95 percent identity in the CDR regions with the CDR regions depicted inthe sequences described above, including CDR regions encoded bycorresponding DNA sequences of plasmids pBW510 and pBW512 as depositedas deposited by Novartis Pharma AG, Forum 1, CH-4002 Bas., Switzerland,in DSMZ on Apr. 29, 2009 with accession number DSM22542 and DSM22543respectively. In some embodiments, it include mutant amino acidsequences wherein no more than 1, 2, 3, 4 or 5 amino acids have beenmutated by amino acid deletion, insertion or substitution in the CDRregions when compared with the CDR regions depicted in the sequencedescribed above.

Further, variable heavy chain parental nucleotide sequences are shown inSEQ ID NO 64. Variable light chain parental nucleotide sequences areshown in SEQ ID NO 57. Full length light chain nucleotide sequencesoptimized for expression in a mammalian cell are shown in SEQ ID NOs83-86. Full length heavy chain nucleotide sequences optimized forexpression in a mammalian cell are shown in SEQ ID NOs 79-82. Otherantibodies of the invention include amino acids or nucleic acids thathave been mutated, yet have at least 60, 70, 80, 90 or 95 percentidentity to the sequences described above. In some embodiments, itinclude mutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5amino acids have been mutated by amino acid deletion, insertion orsubstitution in the variable regions when compared with the variableregions depicted in the sequence described above.

Since each of these antibodies binds the same epitope and are progeniesfrom the same parental antibody, the V_(H), V_(L), full length lightchain, and full length heavy chain sequences (nucleotide sequences andamino acid sequences) can be “mixed and matched” to create otheranti-BAFFR binding molecules of the invention. BAFFR binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed above and in the Examples (e.g., ELISAs). When these chainsare mixed and matched, a V_(H) sequence from a particular V_(H)/V_(L)pairing should be replaced with a structurally similar V_(H) sequence.Likewise a full length heavy chain sequence from a particular fulllength heavy chain/full length light chain pairing should be replacedwith a structurally similar full length heavy chain sequence. Likewise,a V_(L) sequence from a particular V_(H)/V_(L) pairing should bereplaced with a structurally similar V_(L) sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence. Accordingly, inone aspect, the invention provides an isolated recombinant antibody orantigen binding region thereof having: a heavy chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 50-56; and a light chain variable region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 43-49;wherein the antibody specifically binds to BAFFR.

In another aspect, the invention provides

-   -   (i) an isolated recombinant antibody having: a full length heavy        chain comprising an amino acid sequence selected from the group        consisting of SEQ ID NOs:75-78; and a full length light chain        comprising an amino acid sequence selected from the group        consisting of SEQ ID NOs:71-74; wherein the antibody        specifically binds to BAFFR, or    -   (ii) a functional protein comprising an antigen binding portion        thereof.

In another aspect, the invention provides

-   -   (i) an isolated recombinant antibody having: a full length heavy        chain encoded by a nucleotide sequence that has been optimized        for expression in the cell of a mammalian selected from the        group consisting of SEQ ID NOs:79-82; and a full length light        chain encoded by a nucleotide sequence that has been optimized        for expression in the cell of a mammalian selected from the        group consisting of SEQ ID NOs:83-86; wherein the antibody        specifically binds to BAFFR; or,    -   (ii) a functional protein comprising an antigen binding portion        thereof.

The amino acid sequences of the V_(H) CDR1s of the antibodies are shownin SEQ ID NOs: 1-7. The amino acid sequences of the V_(H) CDR2s of theantibodies are shown in SEQ ID NOs: 8-14. The amino acid sequences ofthe V_(H) CDR3s of the antibodies are shown in SEQ ID NOs: 15-21. Theamino acid sequences of the V_(L) CDR1s of the antibodies are shown inSEQ ID NOs: 22-28. The amino acid sequences of the V_(L) CDR2s of theantibodies are shown in SEQ ID NOs: 29-35. The amino acid sequences ofthe V_(L) CDR3s of the antibodies are shown in SEQ ID NOs: 36-42. TheCDR regions set forth in SEQ ID NOs:1-42 are delineated using the Kabatsystem (Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to BAFFR and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, each antibody containing a V_(H)CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 create other anti-BAFFR bindingmolecules of the invention. BAFFR binding of such “mixed and matched”antibodies can be tested using the binding assays described above and inthe Examples (e.g., ELISAs). When V_(H) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(H)sequence should be replaced with a structurally similar CDR sequence(s).Likewise, when V_(L) CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular V_(L) sequence should be replacedwith a structurally similar CDR sequence(s). It will be readily apparentto the ordinarily skilled artisan that novel V_(H) and V_(L) sequencescan be created by substituting one or more V_(H) and/or V_(L) CDR regionsequences with structurally similar sequences from the CDR sequencesshown herein for monoclonal antibodies of the present invention.

In some embodiments, isolated recombinant antibodies, or antigen bindingregions thereof have: a heavy chain variable region CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:1-7; a heavy chain variable region CDR2 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 8-14; a heavychain variable region CDR3 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 15-21; a light chain variableregion CDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22-28; a light chain variable region CDR2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 29-35; and a light chain variable region CDR3 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:36-42; wherein the antibody specifically binds BAFFR.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 2; a heavy chain variable region CDR2 of SEQID NO: 9; a heavy chain variable region CDR3 of SEQ ID NO: 16; a lightchain variable region CDR1 of SEQ ID NO: 23; a light chain variableregion CDR2 of SEQ ID NO: 30; and a light chain variable region CDR3 ofSEQ ID NO: 37.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQID NO: 10; a heavy chain variable region CDR3 of SEQ ID NO: 17; a lightchain variable region CDR1 of SEQ ID NO: 24; a light chain variableregion CDR2 of SEQ ID NO: 31; and a light chain variable region CDR3 ofSEQ ID NO: 38.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 4; a heavy chain variable region CDR2 of SEQID NO: 11; a heavy chain variable region CDR3 of SEQ ID NO: 18; a lightchain variable region CDR1 of SEQ ID NO: 25; a light chain variableregion CDR2 of SEQ ID NO: 32; and a light chain variable region CDR3 ofSEQ ID NO: 39.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 5; a heavy chain variable region CDR2 of SEQID NO: 12; a heavy chain variable region CDR3 of SEQ ID NO: 19; a lightchain variable region CDR1 of SEQ ID NO: 26; a light chain variableregion CDR2 of SEQ ID NO: 33; and a light chain variable region CDR3 ofSEQ ID NO: 40.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQID NO: 13; a heavy chain variable region CDR3 of SEQ ID NO: 20; a lightchain variable region CDR1 of SEQ ID NO: 27; a light chain variableregion CDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 ofSEQ ID NO: 41.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 7; a heavy chain variable region CDR2 of SEQID NO: 14; a heavy chain variable region CDR3 of SEQ ID NO: 21; a lightchain variable region CDR1 of SEQ ID NO: 28; a light chain variableregion CDR2 of SEQ ID NO: 35; and a light chain variable region CDR3 ofSEQ ID NO: 42.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention has full lengthheavy and light chain amino acid sequences; full length heavy and lightchain nucleotide sequences, variable region heavy and light chainnucleotide sequences, or variable region heavy and light chain aminoacid sequences that are homologous to the amino acid and nucleotidesequences of the antibodies described herein, and wherein the antibodiesretain the desired functional properties of the anti-BAFFR antibodies ofthe invention.

For example, the invention provides an isolated recombinant antibody (ora functional protein comprising an antigen binding portion thereof)comprising a heavy chain variable region and a light chain variableregion, wherein: the heavy chain variable region comprises an amino acidsequence that is at least 80%, or at least 90% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 50-56;the light chain variable region comprises an amino acid sequence that isat least 80%, or at least 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 43-49; the antibodyspecifically binds to BAFFR, and the antibody exhibits at least one ofthe following functional properties: it inhibits BLyS induced B cellproliferation, or BLyS induced IgG1 production and it depletes B cell invitro or in vivo.

In a further example, the invention provides an isolated recombinantantibody, (or a functional protein comprising an antigen binding portionthereof) comprising a full length heavy chain and a full length lightchain, wherein: the full length heavy chain comprises an amino acidsequence that is at least 80%, or at least 90% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOs 75-78;the full length light chain comprises an amino acid sequence that is atleast 80%, or at least 90% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs 71-74; the antibody specificallybinds to BAFFR, and the antibody exhibits at least one of the followingfunctional properties: it inhibits BLyS induced B cell proliferation, orBLyS induced IgG1 production and it depletes B cell in vitro or in vivo.

In another example, the invention provides an isolated recombinantantibody (or a functional protein comprising an antigen binding portionthereof), comprising a full length heavy chain and a full length lightchain, wherein: the full length heavy chain is encoded by a nucleotidesequence that is at least 80%, or at least 90% identical to a nucleotidesequence selected from the group consisting of SEQ ID NOs 79-82; thefull length light chain is encoded by a nucleotide sequence that is atleast 80%, or at least 90% identical to a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs 83-86; the antibody specificallybinds to BAFFR, and the antibody exhibits at least one of the followingfunctional properties: it inhibits BLyS induced B cell proliferation, orBLyS induced IgG1 production and it depletes B cell in vitro or in vivo.

In various embodiments, the antibody may exhibit one or more, two ormore, or three of the functional properties discussed above. Theantibody can be, for example, a human antibody, a humanized antibody ora chimeric antibody. Preferably the antibody is a fully human IgG1antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to thesequences set forth above. In other embodiments, the V_(H) and/or V_(L)amino acid sequences may be identical except an amino acid substitutionin no more than 1, 2, 3, 4 or 5 amino acid position. An antibody havingV_(H) and V_(L) regions having high (i. e., 80% or greater) identity tothe V_(H) and V_(L) regions of SEQ ID NOs 50-56 and SEQ ID NOs 43-49respectively, can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NOs:64-70 and 57-63 respectively, followed by testing of the encoded alteredantibody for retained function (i. e., the functions set forth above)using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth above. Anantibody having a full length heavy chain and full length light chainhaving high (i.e., 80% or greater) identity to the full length heavychains of any of SEQ ID NOs 75-78 and full length light chains of any ofSEQ ID NOs 71-74 respectively, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs 79-82 and SEQ ID NOs 83-86 respectively, followed bytesting of the encoded altered antibody for retained function (i. e.,the functions set forth above) using the functional assays describedherein.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth above.

In other embodiments, the variable regions of heavy chain and/or lightchain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequences set forth above

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i. e., % identity=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Com put. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package. usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-BAFFR antibodies of the invention. Accordingly,the invention provides an isolated recombinant antibody, or a functionalprotein comprising an antigen binding portion thereof, consisting of aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencesand a light chain variable region comprising CDR1, CDR2, and CDR3sequences, wherein: the heavy chain variable region CDR1 amino acidsequences are selected from the group consisting of SEQ ID NOs:1-7, andconservative modifications thereof; the heavy chain variable region CDR2amino acid sequences are selected from the group consisting of SEQ IDNOs: 8-14, and conservative modifications thereof; the heavy chainvariable region CDR3 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 15-21, and conservative modifications thereof;the light chain variable regions CDR1 amino acid sequences are selectedfrom the group consisting of SEQ ID NOs: 22-28, and conservativemodifications thereof; the light chain variable regions CDR2 amino acidsequences are selected from the group consisting of SEQ ID NOs: 29-35,and conservative modifications thereof; the light chain variable regionsof CDR3 amino acid sequences are selected from the group consisting ofSEQ ID NOs: 36-42, and conservative modifications thereof; the antibodyspecifically binds to BAFFR, and the antibody exhibits at least one ofthe following functional properties: it inhibits BLyS induced B cellproliferation, or BLyS induced IgG1 production and it depletes B cellsin vitro or in vivo.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

In other embodiments, an antibody of the invention optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the anti-BAFFRantibodies of the invention. Accordingly, the invention provides anisolated monoclonal antibody optimized for expression in a mammaliancell consisting of a full length heavy chain and a full length lightchain wherein: the full length heavy chain has amino acid sequencesselected from the group of SEQ ID NOs: 75-78, and conservativemodifications thereof; and the full length light chain has amino acidsequences selected from the group of SEQ ID NOs: 71-74, and conservativemodifications thereof; the antibody specifically binds to BAFFR; and theantibody exhibits at least one of the following functional properties:it inhibits BLyS induced B cell proliferation, or BLyS induced IgG1production and it depletes B cell in vitro or in vivo.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-BAFFR Antibodies of theInvention

In another embodiment, the invention provides antibodies that bind tothe same epitope as do the various specific anti-BAFFR antibodies of theinvention described herein. All the antibodies described in the Examplesthat are capable of blocking the BLyS induced effect bind the sameepitope in BAFFR with high affinity, said epitope being comprisedbetween amino acids of SEQ ID NO:88.

Additional antibodies can therefore be identified based on their abilityto cross-compete (e.g., to competitively inhibit the binding of, in astatistically significant manner) with other antibodies of the inventionin standard BAFFR binding assays. The ability of a test antibody toinhibit the binding of antibodies of the present invention to humanBAFFR demonstrates that the test antibody can compete with that antibodyfor binding to human BAFFR; such an antibody may, according tonon-limiting theory, bind to the same or a related (e.g., a structurallysimilar or spatially proximal) epitope on human BAFFR as the antibodywith which it competes. Thus, another aspect of the invention providesantibodies that bind to the same antigen as, and compete with, theantibodies disclosed herein by sequence. In a certain embodiment, theantibody that binds to the same epitope on human BAFFR as the antibodiesof the present invention is a human recombinant antibody. Such humanrecombinant antibodies can be prepared and isolated as described in theExamples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal anti-BAFFR antibody, or a functional protein comprising anantigen binding portion thereof, comprising a heavy chain variableregion comprising CDR1 sequences having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-7; CDR2 sequences having anamino acid sequence selected from the group consisting of SEQ ID NOs:8-14; CDR3 sequences having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15-21, respectively; and a light chainvariable region having CDR1 sequences having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22-28; CDR2 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 29-35; and CDR3 sequences consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 36-42, respectively.Thus, such antibodies contain the V_(H) and V_(L) CDR sequences ofmonoclonal antibodies, yet may contain different framework sequencesfrom these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at wwvv.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedanti-BAFFR monoclonal antibodies, or a functional protein comprising anantigen binding portion thereof, consisting of a heavy chain variableregion having: a V_(H) CDR1 region consisting of an amino acid sequenceselected from the group having SEQ ID NOs: 1-7 or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 1-7; a V_(H) CDR2 region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:8-14, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 8-14; a V_(H) CDR3 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 15-21, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 15-21; a V_(L) CDR1region having an amino acid sequence selected from the group consistingof SEQ ID NOs: 22-28, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 22-28; a V_(L) CDR2 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 29-35, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 29-35;and a V_(L) CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 36-42, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 36-42.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to BAFFR. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof (such as those disclosed elsewhere herein), andinclude immunoglobulins of other animal species, preferably havinghumanized aspects. Single heavy-chain antibodies such as thoseidentified in camelids are of particular interest in this regard. Novelframeworks, scaffolds and fragments continue to be discovered anddeveloped by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target protein of SEQ ID NO: 87. Suchcompounds are known herein as “polypeptides comprising a target-specificbinding region”. Examples of non-immunoglobulin framework are furtherdescribed in the sections below (camelid antibodies and non-antibodyscaffold).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. etal., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 BioconjugateChem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineeredlibraries of camelid antibodies and antibody fragments are commerciallyavailable, for example, from Ablynx, Ghent, Belgium. As with otherantibodies of non-human origin, an amino acid sequence of a camelidantibody can be altered recombinantly to obtain a sequence that moreclosely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies tohumans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for BAFFR. In certain embodiments herein,the camelid antibody or nanobody is naturally produced in the camelidanimal, i.e., is produced by the camelid following immunization withBAFFR or a peptide fragment thereof, using techniques described hereinfor other antibodies. Alternatively, the anti-BAFFR camelid nanobody isengineered, i.e., produced by selection for example from a library ofphage displaying appropriately mutagenized camelid nanobody proteinsusing panning procedures with BAFFR as a target as described in theexamples herein. In one embodiment, an antibody of the disclosure iscamelized, having a camelid framework and V_(H) CDR1, CDR2 and/or CDR3regions as disclosed herein. Engineered nanobodies can further becustomized by genetic engineering to have a half life in a recipientsubject of from 45 minutes to two weeks. In a specific embodiment, thecamelid antibody or nanobody is obtained by grafting the CDRs sequencesof the heavy or light chain of the human antibodies of the inventioninto nanobody or single domain antibody framework sequences, asdescribed for example in PCT/EP93/02214.

Non-Antibody Scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, Adnectins (fibronectin) (Compound Therapeutics, Inc.,Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(i) Fibronectin Scaffold

The fibronectin scaffolds are based preferably on fibronectin type IIIdomain (e.g., the tenth module of the fibronectin type III (10 Fn3domain)). The fibronectin type III domain has 7 or 8 beta strands whichare distributed between two beta sheets, which themselves pack againsteach other to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

(ii) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(ii) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example,20040175756; 20050053973; 20050048512; and 20060008844.

(vi) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(v) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris Brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT WO 199916873.

(vi) Affilin—Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368

(vii) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa)mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody.

Typically such framework modifications are made to decrease theimmunogenicity of the antibody. For example, one approach is to“backnnutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. To return theframework region sequences to their germline configuration, the somaticmutations can be “backmutated” to the germline sequence by, for example,site-directed mutagenesis or PCR-mediated mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et at.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγreceptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the antigen. Suchcarbohydrate modifications can be accomplished by; for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated ornon-fucosylated antibody having reduced amounts of or no fucosylresidues or an antibody having increased bisecting GlcNac structures.Such altered glycosylation patterns have been demonstrated to increasethe ADCC ability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. For example, EP1,176,195 by Hang et al. describes a cell line with a functionallydisrupted FUT8 gene, which encodes a fucosyl transferase, such thatantibodies expressed in such a cell line exhibit hypofucosylation or aredevoid of fucosyl residues. Therefore, in one embodiment, the antibodiesof the invention are produced by recombinant expression in a cell linewhich exhibit hypofucosylation or non-fucosylation pattern, for example,a mammalian cell line with deficient expression of the FUT8 geneencoding fucosyltransferase. PCT Publication WO 03/035835 by Prestadescribes a variant CHO cell line, Lecl3 cells, with reduced ability toattach fucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al., 2002 J. Bioi. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).Eureka Therapeutics further describes genetically engineered CHOmammalian cells capable of producing antibodies with altered mammalianglycosylation pattern devoid of fucosyl residues. Alternatively, theantibodies of the invention can be produced in yeasts or filamentousfungi engineered for mammalian-like glycosylation pattern and capable ofproducing antibodies lacking fucose as glycosylation pattern (see forexample EP1297172B1).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.The pegylation can be carried out by an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by theinvention is a conjugate or a protein fusion of at least theantigen-binding region of the antibody of the invention to serumprotein, such as human serum albumin or a fragment thereof to increasehalf-life of the resulting molecule. Such approach is for exampledescribed in Ballance et al. EP0322094.

Another possibility is a fusion of at least the antigen-binding regionof the antibody of the invention to proteins capable of binding to serumproteins, such human serum albumin to increase half life of theresulting molecule. Such approach is for example described in Nygren etal., EP 0 486 525.

Methods of Engineering Altered Antibodies

As discussed above, the anti-BAFFR antibodies having V_(H) and V_(L)sequences or full length heavy and light chain sequences shown hereincan be used to create new anti-BAFFR antibodies by modifying full lengthheavy chain and/or light chain sequences, V_(H) and/or V_(L) sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of an anti-BAFFR antibody of theinvention are used to create structurally related anti-BAFFR antibodiesthat retain at least one functional property of the antibodies of theinvention, such as binding to human BAFFR and also inhibiting one ormore functional properties of BAFFR (e.g., antagonistic activity, B celldepleting activity).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-BAFFR antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-BAFFR antibody consisting of: a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 1-7, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 8-14 and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 15-21; and a light chain variable regionantibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 22-28, a CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 29-35 and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 36-42; altering at least one amino acidresidue within the heavy chain variable region antibody sequence and/orthe light chain variable region antibody sequence to create at least onealtered antibody sequence; and expressing the altered antibody sequenceas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-BAFFR antibody optimized for expression in a mammaliancell consisting of: a full length heavy chain antibody sequence having asequence selected from the group of SEQ ID NOs: 75-78; and a full lengthlight chain antibody sequence having a sequence selected from the groupof 71-74; altering at least one amino acid residue within the fulllength heavy chain antibody sequence and/or the full length light chainantibody sequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences selected among the groupconsisting of SEQ ID NO:15-21 and SEQ ID NO: 36-42 or minimal essentialbinding determinants as described in US20050255552 and diversity on CDR1and CDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the anti-BAFFR antibodies described herein,which functional properties include, but are not limited to,specifically binding to human BAFFR; and/or it inhibits BLyS induced Bcell proliferation, BLyS induced B or BLyS induced IgG1 production;and/or depletes human B cell in vitro or in vivo.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-BAFFR antibody coding sequence and the resultingmodified anti-BAFFR antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. Examples of full length lightchain nucleotide sequences optimized for expression in a mammalian cellare shown in SEQ ID NOs:83-86. Examples of full length heavy chainnucleotide sequences optimized for expression in a mammalian cell areshown in SEQ ID NOs: 79-82.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromvarious phage clones that are members of the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to an scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA molecule, or to a fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked”, as used in this context, isintended to mean that the two DNA fragments are joined in a functionalmanner, for example, such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame, or such that the protein is expressedunder control of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments,the heavy chain constant region is selected among IgG1 isotypes. For aFab fragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., 1988 Science 242:423-426; Huston et at., 1988 Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

Generation of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstBAFFR can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851). See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92103918, WO 93/12227, WO 94/25585, WO 97113852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-BAFFR antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-BAFFR antibodies of the invention. For example, mice carrying botha human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise anti-BAFFRantibodies of the invention.

Human recombinant antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner etal.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat.Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×145in flat bottom microtiter plates, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0:055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the CH segment(s) within the vector andthe V_(L) segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, inparticular mammalian host cells, is discussed because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today6:12-13).

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myelomacells, COS cells and SP2 cells). In particular, for use with NSO myelomacells, another expression system is the GS gene expression system shownin WO 87/04462, WO 89/01036 and EP 338,841. In one embodiment, mammalianhost cells for expressing the recombinant antibodies of the inventioninclude mammalian cell lines deficient for FUT8 gene expression, forexample as described in U.S. Pat. No. 6,946,292B2. When recombinantexpression vectors encoding antibody genes are introduced into mammalianhost cells, the antibodies are produced by culturing the host cells fora period of time sufficient to allow for expression of the antibody inthe host cells or secretion of the antibody into the culture medium inwhich the host cells are grown. Antibodies can be recovered from theculture medium using standard protein purification methods.

Immunoconjugates

In another aspect, the present invention features an anti-BAFFRantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thioepachloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody of the invention include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg™;Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.,2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al., 2003 CancerImmunol. Immunother. 52:328-337; Payne, G., 2003 Cancer Cell 3:207-212;Allen, T. M., 2002 Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman,R. J., 2002 Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P. D. andSpringer, C. J., 2001 Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine^(I31),indium¹¹¹, yttrium⁹⁰, and lutetium¹⁷⁷. Method for preparingradioimmunconjugates are established in the art. Examples ofradioimmunoconjugates are commercially available, including Zevalin™(DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similarmethods can be used to prepare radioimmunoconjugates using theantibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et at., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific ormultispecific molecules comprising an anti-BAFFR antibody, or a fragmentthereof, of the invention. An antibody of the invention, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the invention, an antibody of the invention canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for BAFFR and a secondbinding specificity for a second target epitope. For example, the secondtarget epitope is another epitope of BAFFR different from the firsttarget epitope. Another example is a bispecific molecule comprising atleast one first binding specificity for BAFFR and a second bindingspecificity for an epitope within CD20.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccininnidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mA×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present invention provides multivalent antibodiescomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to BAFFR. In one embodiment,the multivalent antibodies provides at least two, three or fourantigen-binding portions of the antibodies. The antigen-binding portionscan be linked together via protein fusion or covalent or non covalentlinkage. Alternatively, methods of linkage have been described for thebispecific molecules. Tetravalent compounds can be obtained for exampleby cross-linking antibodies of the antibodies of the invention with anantibody that binds to the constant regions of the antibodies of theinvention, for example the Fc or hinge region.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodiesthat bind to different epitopes on the target antigen or that havecomplementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-BAFFR antibody of the presentinvention combined with at least one other anti-inflammatory or anotherchemotherapeutic agent, for example, a cytotoxic, anti-cancer oranti-proliferative agent. Examples of therapeutic agents that can beused in combination therapy are described in greater detail below in thesection on uses of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediannine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Reviews on the development of stable protein (e.g., antibody)formulations may be found in Cleland et al. (1993) Crit. Reviews. Ther.Drug Carrier Systems 10(4):307-377 and Wei Wang (1999) Int. J.Pharmaceutics 185:129-88. Additional formulation discussions forantibodies may be found, e.g., in Daugherty and Mrsny (2006) AdvancedDrug Delivery Reviews 58: 686-706; U.S. Pat. No. 6,171,586; U.S. Pat.No. 4,618,486; US20060286103; WO06044908; WO07095337; WO04016286;Colandene et al. (2007) J. Pharm. Sci 96: 1598-1608; Schulman (2001) Am.J. Respir. Crit. Care Med. 164:S6-S11 and other known references.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Sterile injectable solutions can be prepared by incorporating theantibody of the invention in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

When a therapeutically effective amount of an antibody of the inventionis administered by, e.g., intravenous, cutaneous or subcutaneousinjection, the binding agent will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein solutions, having due regard to pH,isotonicity, stability, and the like, is within the skill in the art. Apreferred pharmaceutical composition for intravenous, cutaneous, orsubcutaneous injection should contain, in addition to binding agents, anisotonic vehicle such as sodium chloride injection, Ringer's injection,dextrose injection, dextrose and sodium chloride injection, lactatedRinger's injection, or other vehicle as known in the art. Thepharmaceutical composition(s) of the present disclosure may also containstabilizers, preservatives, buffers, antioxidants, or other additiveknown to those of skill in the art.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, from about 0.1percent to about 70 percent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals. For administration of theantibody, the dosage ranges from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg, of the host body weight. For example dosagescan be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight,5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg. An exemplary treatment regime entails administration once perweek, once every two weeks, once every three weeks, once every fourweeks, once a month, once every 3 months or once every three to 6months. Dosage regimens for an anti-BAFFR antibody of the inventioninclude 1 mg/kg body weight or 3 mg/kg body weight by intravenousadministration, with the antibody being given using one of the followingdosing schedules: every four weeks for six dosages, then every threemonths; every three weeks; 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-BAFFR antibody of theinvention can results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.

A composition of the present invention can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the inventioninclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastennal injection andinfusion.

Alternatively, an antibody of the invention can be administered by anonparenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. Many other such implants, deliverysystems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade,1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M.Owais et al., 1995 Antimicrob. Agents Chernother. 39:180); surfactantprotein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen, 1994 FEBSLett. 346:123; J. J. Killion; I. J. Fidler,1994 Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies of the present invention have in vitro and in vivodiagnostic and therapeutic utilities. For example, these molecules canbe administered to cells in culture, e.g. in vitro or in vivo, or in asubject, e.g., in vivo, to treat, prevent or diagnose a variety ofdisorders. The term “subject” as used herein is intended to includehuman and non-human animals. Non-human animals include all vertebrates,e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, and reptiles.

The methods are particularly suitable for treating, preventing ordiagnosing BAFFR-related disorders and/or autoimmune diseases, e.g.,systemic lupus erythematosus or rheumatoid arthritis.

The invention also provides methods for depleting B cells in an animal,preferably depleting or killing human B cell by administering acomposition comprising a therapeutically efficient dose of theantibodies of the invention.

As used herein, “a BAFFR-related disorder” includes conditionsassociated with or characterized by aberrant BLyS levels and/or diseasesor conditions that can be treated by depleting or killing B cells. Theseinclude inflammatory conditions, allergies and allergic conditions,hypersensitivity reactions, autoimmune diseases, severe infections, andorgan or tissue transplant rejection. These further include B-cellneoplasms.

For example, the antibodies of the invention may be used for thetreatment of recipients of heart, lung, combined heart-lung, liver,kidney, pancreatic, skin or corneal transplants, including allograftrejection or xenograft rejection, and for the prevention ofgraft-versus-host disease, such as following bone marrow transplant, andorgan transplant associated arteriosclerosis.

The antibodies of the invention are useful for the treatment,prevention, or amelioration of autoimmune disease and of inflammatoryconditions, in particular inflammatory conditions with an aetiologyincluding an autoimmune component such as arthritis (for examplerheumatoid arthritis, arthritis chronica progrediente and arthritisdeformans) and rheumatic diseases, including inflammatory conditions andrheumatic diseases involving bone loss, inflammatory pain,spondyloarhropathies including ankolsing spondylitis, Reiter syndrome,reactive arthritis, psoriatic arthritis, and enterophathics arthritis,hypersensitivity (including both airways hypersensitivity and dermalhypersensitivity) and allergies. Specific auto-immune diseases for whichantibodies of the invention may be employed include autoimmunehaematological disorders (including e.g. hemolytic anaemia, aplasticanaemia, pure red cell anaemia and idiopathic thrombocytopenia),acquired hemophilia A, cold agglutinin disease, cryoglobulinemia,thrombotic thrombocytopenic purpura, Sjögren's syndrome, systemic lupuserythematosus, inflammatory muscle disorders, polychondritis,sclerodoma, anti-neutrophil cytoplasmic antibody-associated vasculitis,IgM mediated neuropathy, opsoclonus myoclonus syndrome, Wegenergranulomatosis, dermatomyositis, chronic active hepatitis, myastheniagravis, psoriasis, Steven-Johnson syndrome, pemphigus vulgaris,pemphigus foliacius, idiopathic sprue, autoimmune inflammatory boweldisease (including e.g. ulcerative colitis, Crohn's disease andIrritable Bowel Syndrome), endocrine ophthalmopathy, Graves' disease,sarcoidosis, multiple sclerosis, neuromyelitis optica, primary biliarycirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis(anterior, intermediate and posterior as well as panuveitis),keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, psoriatic arthritis and glomerulonephritis (with andwithout nephrotic syndrome, e.g. including idiopathic nephrotic syndromeor minimal change nephropathy), tumors, inflammatory disease of skin andcornea, myositis, loosening of bone implants, metabolic disorders, suchas atherosclerosis, diabetes, and dislipidemia.

The antibodies of the invention are also useful for the treatment,prevention, or amelioration of asthma, bronchitis, pneumoconiosis,pulmonary emphysema, and other obstructive or inflammatory diseases ofthe airways.

The antibodies of the invention are also useful for treating diseases ofbone metabolism including osteoarthritis, osteoporosis and otherinflammatory arthritides, and bone loss in general, includingage-related bone loss, and in particular periodontal disease.

Since BAFFR binding to human peripheral blood lymphocytes and to thehuman B-cell line is mediated by BAFFR polypeptides, the antibodies ofthe invention may also be useful in diagnosing or treating B-cellneoplasms. Examples of such diseases and conditions include, but are notlimited to, B-cell Non-Hodgkin's lymphomas, such as small lymphocyticlymphoma, lymphoplasmacytoid lymphoma, mantle cell lymphoma, follicularlymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large celllymphoma, and Burkitt's lymphoma; precursor B-lymphoblastic leukemia;and B-cell chronic lymphocytic leukemia, and multiple myeloma. OtherB-cell neoplasms are encompassed within the scope of the invention.

The antibodies of the invention may be administered as the sole activeingredient or in conjunction with, e.g. as an adjuvant to or incombination to, other drugs e.g. immunosuppressive or immunomodulatingagents or other anti-inflammatory agents or other cytotoxic oranti-cancer agents, e.g. for the treatment or prevention of diseasesmentioned above. For example, the antibodies of the invention may beused in combination with DMARD, e.g. Gold salts, sulphasalazine,antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolicacid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide,glucocorticoids; a calcineurin inhibitor, e.g. cyclosporin A or FK 506;a modulator of lymphocyte recirculation, e.g. FTY720 and FTY720 analogs;an mTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxyethyl)-rapamycin,CCl779, ABT578, AP23573 or TAFA-93; an ascomycin havingimmuno-suppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclo-phos-phamide; azathioprene; methotrexate;mizoribine; mycophenolic acid; myco-pheno-late mofetil;15-deoxyspergualine or an immunosuppressive homologue, analogue orderivative thereof; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD8, CD11a, CD25, CD28, CD40. CD45, CD52, CD58, CD80, CD86 or theirligands; other immunomodulatory compounds, e.g. a recombinant bindingmolecule having at least a portion of the extracellular domain of CTLA4or a mutant thereof, e.g. an at least extracellular portion of CTLA4 ora mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4Ig(for ex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y;adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3antagonists, VCAM-4 antagonists or VLA-4 antagonists; or achemotherapeutic agent, e.g. paclitaxel, gemcitabine, cisplatinum,doxorubicin or 5-fluorouracil; anti TNF agents, e.g. monoclonalantibodies to TNF, e.g. infliximab, adalimumab, CDP870, or receptorconstructs to TNF-RI or TNF-RII, e.g. Etanercept, PEG-TNF-RI; blockersof proinflammatory cytokines, IL-1 blockers, e.g. Anakinra or IL-1 trap,AAL160, ACZ 885, IL-6 blockers; chemokines blockers, e.g inhibitors oractivators of proteases, e.g. metalloproteases, anti-IL4 antibodies,anti-IL-15 antibodies, anti-IL-6 antibodies, anti-IL-21 antibodies,anti-IL-12 antibodies, anti-p40 antibodies, anti-IL-17 antibodies,anti-CD20 antibodies, NSAIDs, such as aspirin or an anti-infectiousagent (list not limited to the agent mentioned).

In accordance with the foregoing the present invention provides in a yetfurther aspect:

A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount of aBAFFR antagonist, e.g., an antibody of the invention, and at least onesecond drug substance, said second drug substance being aimmuno-suppressive/immunomodulatory, anti-inflammatory chemotherapeuticor anti-infectious drug, e.g. as indicated above.

Or, a therapeutic combination, e.g. a kit, comprising of atherapeutically effective amount of a) an BAFFR antagonist, e.g. anantibody of the invention, and b) at least one second substance selectedfrom a immuno-suppressive/immunomodulatory, anti-inflammatorychemotherapeutic or anti-infectious drug, e.g. as indicated above. Thekit may comprise instructions for its administration.

Where the antibodies of the invention are administered in conjunctionwith other immuno-suppressive/immunomodulatory, anti-inflammatorychemotherapeutic or anti-infectious therapy, dosages of theco-administered combination compound will of course vary depending onthe type of co-drug employed, e.g. whether it is a DMARD, anti-TNF, IL-1blacker or others, on the specific drug employed, on the condition beingtreated and so forth.

In one specific embodiment, the antibodies of the invention may beadministered in combination with another B-cell killing agent, i.e.,e.g, a CD20 targeting antibody with ADCC activity, such as Rituximab.

In other embodiment, the antibodies of the invention are administeredonly to patient population which is selected among patients sufferingfrom SLE or RA and exhibiting an abnormal serum level of BLyS. In otherembodiment, the antibodies of the invention are administered only topatient population which are selected among group of patients whichrespond to anti-BLyS treatment. Biomarkers that identify patients thathave an increased likelihood of responding to anti-BLyS treatment may beany of the following without being limited to these: elevated levels ofserum BLyS, elevated levels of certain B cell subsets, presence orabsence of certain types of auto-antibodies.

In one embodiment, the antibodies of the invention can be used to detectlevels of BAFFR, or levels of cells that contain BAFFR. This can beachieved, for example, by contacting a sample (such as an in vitrosample) and a control sample with the anti-BAFFR antibody underconditions that allow for the formation of a complex between theantibody and BAFFR. Any complexes formed between the antibody and BAFFRare detected and compared in the sample and the control. For example,standard detection methods, well known in the art, such as ELISA andflow cytometic assays, can be performed using the compositions of theinvention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of BAFFR (e.g., human BAFFR antigen) in a sample,or measuring the amount of BAFFR, comprising contacting the sample, anda control sample, with an antibody of the invention, or an antigenbinding region thereof, which specifically binds to BAFFR, underconditions that allow for formation of a complex between the antibody orportion thereof and BAFFR. The formation of a complex is then detected,wherein a difference in complex formation between the sample compared tothe control sample is indicative of the presence of BAFFR in the sample.

Also within the scope of the invention are kits consisting of thecompositions (e.g., antibodies, human antibodies and bispecificmolecules) of the invention and instructions for use. The kit canfurther contain a least one additional reagent, or one or moreadditional antibodies of the invention (e.g., an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit. The kit may further comprisetools for diagnosing whether a patient belongs to a group that respondsto an anti-BAFFR antibody treatment, as defined above.

The invention having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of Collagen-induced arthritis (CIA) in DBA/1mice (MCOL37) after immunization at day 0, Booster at day 21, andTreatment starting day minus 9. The square symbols show the paws scoringfor Anti-BAFF Ab-MOR6743-mIgG2a with a dose equal to 200 μg/200 μl i.p.2× a week, (n=8). The triangle symbols shows the paws scoring for anIsotype Anti CSA-IgG2a control with a dose equal to 200 μg/200 μl i.p.2× a week, (n=7). Statistics: Dunnett Multiple Comparisons Test (One-wayANOVA). Compared with the Vehicle control (p<0.05*,p<0.01**,p>0.05n.s.).

FIG. 2 shows the absolute number of CD20+ and CD40+ peripheral bloodcells in 3 cynomolgous monkeys after 20 mg/kg intravenous administrationof anti-BAFFR (MOR06654) antibodies.

FIG. 3A shows the absolute number of CD20+ and CD40+ peripheral bloodcells in 3 cynomolgous monkeys after 20 μg/kg intravenous administrationof anti-BAFFR (MOR06654) antibodies.

FIG. 3B shows the absolute number o CD20+ and CD40+ peripheral bloodcells in 3 cynomolgous monkeys after 20 μg/kg intravenous administrationof non-fucosylated anti-BAFFR (MOR06654) antibodies.

EXPERIMENTAL PARTS

1. Screening Assays

FACS Screening of Initial Pannings

For the detection of BAFFR binding Fab antibodies from first primaryscreening, e.g., phage display screening and/or for the secondaryscreening process of ELISA positive clones, lysates of selected E. coliclones can be screened in FACS as follows:

Cells of the respective cell line (parental cells or cells transfectedwith BAFFR) are counted and adjusted to 2×10⁷ cells/ml in PBS/3%FCS/0.02% NaN₃ (FACS buffer). In a 96-well round bottom plate, 2×10⁵cells are mixed with 35 μl Fab-containing 1bacterial lysate in a finalvolume of 100 μl FACS buffer and incubated on a shaker at 4° C. for onehour. Cells are then washed once with FACS buffer and resuspended inphycoerythrin-conjugated goat anti-human IgG secondary antibody whichhas been diluted 1:200 in FACS buffer. After one hour incubation at 4°C. on a shaker, cells were again washed once with FACS buffer,resuspended in FACS buffer and cell surface binding is measured, forexample, via fluorescence intensity of the cells in the FACSArrayinstrument (Becton Dickinson).

Fc Capture ELISA

To identify BAFFR binding Fab antibodies in the enriched clones fromphage display, lysates of selected E. coli clones are screened in an Fccapture ELISA setting as follows: Maxisorp 384-well plates are coatedwith 20 μg/ml goat anti-human IgG Fc diluted in PBS overnight at 4° C.On the next day, the plates are washed, blocked with 5% MTBST, andincubated with 5 μg/ml BAFFR:Fc (Alexis) for one hour at roomtemperature. In parallel, the Fab containing bacterial lysates areblocked with a final concentration of 2.5% milk powder. Then,pre-blocked bacterial lysates are added to the captured BAFFR:Fc on theplates. Subsequently BAFFR:Fc binding Fabs are detected by incubationwith alkaline phosphatase conjugated goat anti-human IgG, Fab specific,diluted 1:5000 in 0.5% MPBST, followed by addition of AttoPhosfluorescence substrate (Roche Diagnostics). Fluorescence emission at 535nm is recorded with excitation at 430 nm in a TECAN Spectrafluor platereader.

Fab Capture ELISA

For the detection of BAFFR binding Fab antibodies from phage display,lysates of selected E. coli clones are screened in a Fab capture ELISAsetting: Maxisorp 384-well plates are coated with a sheep anti-humanIgG, Fd fragment specific antibody diluted 1:1000 in PBS overnight at 4°C. On the next day, the plates are washed and blocked with TBS/0.05%Tween/5% milk powder (5% MTBST) for one hour at room temperature. Inparallel, the Fab containing bacterial lysates are blocked with a finalconcentration of 2.5% milk powder. Then, pre-blocked bacterial lysatesare added to the capture antibody immobilized on the plates.Subsequently the captured HuCAC-Fab fragments are allowed to bind to 1μg/ml biotinylated BAFFR:Fc (diluted in PBST) which is finally detectedby incubation with Streptavidin conjugated to alkaline phosphatase(Zymax), diluted 1:3000 in 2.5% MPBST, followed by addition of AttoPhosfluorescence substrate (Roche Diagnostics). Fluorescence emission at 535nm was recorded with excitation at 430 nm in a TECAN Spectrafluor platereader.

BAFFR-BLyS Binding ELISA

To directly identify inhibitory antibodies, FLAG-tagged Fabs arescreened in a BAFFR-BLyS binding ELISA: MaxiSorp 96-well plates arecoated with 1 μg/ml hsBLyS in PBS, overnight at 4° C. On the next day,the plates are washed and blocked with PBS/2% BSA for at least one hourat room temperature. In parallel, the Fab containing bacterial lysatesare blocked with a final concentration of 2.5% BSA. The pre-blockedbacterial lysates were incubated with 5 μg/ml monoclonal anti-FLAG M2antibody for 30 min in order to increase the inhibitory activity bycross-linking and subsequently 10 ng/ml biotinylated BAFFR:Fc (dilutedin PBS/2% BSA) are added for another 30 min at room temperature,slightly shaking. The pre-incubated lysate/bio-BAFFR:Fc mixture is addedto plate-bound hsBLyS. After 30 min at room temperature and washing, theBLyS bound bio-BAFFR:Fc is detected by incubation with Streptavidinconjugated to alkaline phosphatase (Zymax) diluted 1:3000 in PBS/2.5%BSA, followed by addition of AttoPhos fluorescence substrate (RocheDiagnostics). Fluorescence emission at 535 nm was recorded withexcitation at 430 nm in a TECAN Spectrafluor plate reader.

In case purified Fabs are used instead of bacterial lysates, theanti-FLAG cross-linking step is omitted.

FACS Screening After Maturation

Second Maturation:

For the screening of binders from the maturation the FACS analysis canbe performed as described with the following changes: the lysate of theselected E. coli clones is diluted until maximum signals are belowsaturation. The lysates are analyzed for binding to Raji cells. 5×10⁴cells per 96-well are mixed with 100 μl of the different dilutedbacterial lysates. The detection of the cell-bound Fabs is performed asdescribed. Clones can be ranked according to their signal strength.

2. Affinity Determination of Antibodies Identified From Screening Assays

Solution Equilibrium Titration (SET) Method for KD Determination UsingBioVeris

Affinity determination in solution can be basically performed asdescribed in the literature (Friquet et al. (1985) J. Immunol. Meth. 77,305-319 and Haenel et al. (2005) Anal. Biochem. 339, 182-184). In orderto improve the sensitivity and accuracy of the SET method, the method istransferred from classical ELISA to ECL based BioVeris technology. 1mg/ml goat-anti-human (Fab)₂ or goat-anti-mouse IgG, Fc fragmentspecific antibodies (Dianova) were labelled with BV-tag™ NHS-Ester(Bioveris Europe, Witney, Oxfordshire, UK) according to themanufacturer's instructions. The experiment is carried out inpolypropylene microliter plates and PBS pH 7.4 supplemented with 0.5%BSA and 0.02% Tween 20 as assay buffer. Unlabeled BAFFR:Fc is diluted in2n series. Wells without antigen were used to determine Smax values.After addition of 100 pM Fab (final concentration in 75 μl finalvolume), the mixture was incubated for 2 hours at RT. Subsequently amixture of 25 μl Dynabeads (0.4 mg/ml M-280 Streptavidin, DYNAL,Hamburg), coated with 0.25 μg/ml biotinylated BAFFR:Fc antigen andBV-tag labeled detection antibody in a final dilution of 1:4000 foranti-human Fab or 1:2000 for anti-mouse IgG were added per well. Afterincubation for 30 min on an Eppendorf shaker (700 rpm) at RT,electrochemiluminescence signals are detected using a M-384 SERIES®Workstation (Bioveris Europe).

Biacore K_(D) Determination on Directly Coated Antigen

The kinetic constants k_(on) and k_(off) are determined with serialdilutions of the respective Fab binding to either Fc-captured BAFFR:Fcor to covalently immobilized BAFFR:Fc (Alexis) or BAFFR (Peprotech)using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden). Forcovalent immobilization of the antigens or the anti-Fc capture antibodystandard EDC-NHS amine coupling chemistry is used. Kinetic measurementswere done in PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄pH 7.4) at a flow rate of 20 μl/min using Fab concentrations rangingfrom 1.5 to 500 nM. The injection time for each concentration is 1 min,followed by either 3 or 15 min dissociation phase. For regeneration twoinjections of Glycine/HCl pH 1.5 are applied. All sensograms are fittedusing the BIA evaluation software 3.1 (Biacore).

For affinity determination in presence of 10% human serum the kineticmeasurement is performed in PBS containing 10% human serum which wassterile filtered (pore size 1.2 and 0.2 μm) prior to use in order toremove protein aggregates after thawing.

Determination of IC₅₀ Values in the BAFFR-BLyS Binding Assay(Competition FACS)

The BAFFR-BLyS binding assay is performed using Raji cells expressingendogenous BAFFR. The BAFFR specific Fabs are used in final dilutionsranging from 40 to 0.001 nM.

The diluted Fabs are incubated in 96-well plates with 5×10⁴ Raji cellsper well for 1 h at 4° C. on a shaker. Then biotinylated hsBLyS is addedin a final concentration of 25 ng/ml and the cells were incubated for 30min at 4° C. on a shaker. The cells are washed twice with FACS bufferand then resuspended in phycoerythrin-conjugated Streptavidin (Dianova)which has been diluted 1:200 in FACS buffer. The staining is performedfor one hour at 4° C. on a shaker. Finally the cells are washed twicewith FACS buffer, resuspended in FACS buffer and BLyS binding to cellsurface BAFFR is detected via fluorescence cytometry in the FACSArrayinstrument (Becton Dickinson).

Determination of IC₅₀ Values in the BAFFR:Fc-BLyS Binding Assay(Competition ELISA)

96-well MaxiSorp plates are coated over night at 4° C. with humansoluble BLyS. After coating, the wells were washed 4 times withPBS/0.05% Tween20 (PBST) and then blocked for 1 h at 37° C. with PBSTcontaining 1% bovine serum albumin followed by 4 washing steps withPBST. Biotinylated human BAFFR:Fc fusion protein (Axxora) at 20 ng/mlwas added and captured for 1 h at 37° C. together with increasingconcentrations of anti-BAFFR antibodies. After another washing round,ExtrAvidin-Alkaline Phosphatase (Sigma) was added to the wells andincubated for 30 minutes at 37° C. The bound phosphatase was detected byadding a solution containing p-nitrophenyl phosphate in diethanolamin(Sigma). The color reaction was stopped after ca. 20 minutes with anequal volume of 2N sodium hydroxide and the absorbance was measured at405 nm on a plate reader (SpectraMax 190, Molecular Devices).

Determination of IC50 Values in the BAFFR Peptide (miniBR3)-BLyS BindingAssay (Competition ELISA)

96-well MaxiSorp plates are coated over night at 4° C. with humansoluble BLyS. After coating, the wells were washed 4 times withPBS/0.05% Tween20 (PBST) and then blocked for 1 h at 37° C. with PBSTcontaining 1% bovine serum albumin followed by 4 washing steps withPBST. Biotinylated BAFFR derived peptide (miniBR3, PiCHEM, Austria) at20 ng/ml was added and captured for 1 h at 37° C. together withincreasing concentrations of anti-BAFFR antibodies. After anotherwashing round, ExtrAvidin-Alkaline Phosphatase (Sigma) was added to thewells and incubated for 30 minutes at 37° C. The bound phosphatase wasdetected by adding a solution containing p-nitrophenyl phosphate indiethanolamin (Sigma). The color reaction was stopped after ca. 20minutes with an equal volume of 2N sodium hydroxide and the absorbancewas measured at 405 nm on a plate reader (SpectraMax 190, MolecularDevices).

Analysis of Cross-Reactivity to Cynomolgus BAFFR

The cross-reactivity of the Fabs to cynomolgus BAFFR is tested in FACStitration analysis on cyno BAFFR transfected HEK293T cells. The finalcandidate Fabs are used in dilutions ranging from 177 nM to 0.001 nM.The diluted Fabs are incubated in 96-well plates with 5×10⁴ cells perwell for 1 h at 4° C. on a shaker. Then the cells are washed twice withFACS buffer and resuspended in phycoerythrin-conjugated goat anti-humanIgG (Dianova) which has been diluted 1:200 in FACS buffer. The stainingis performed for 1 h at 4° C. on a shaker. Finally, the cells are washedtwice with FACS buffer and Fab binding is detected via fluorescencecytometry in the FACSArray instrument (Becton Dickinson).

Analysis of Cross-Reactivity to BCMA and TACI

FACS

The cross-reactivity of the Fabs to BCMA is tested in FACS titrationanalysis on BCMA transfected HEK293T cells: the Fabs are used in finalconcentrations ranging from high nM or even μM down to pM. The stainingof the cells and the detection of Fab binding are performed asdescribed.

Elisa

384-well MaxiSorp plates are coated over night at 4° C. with a goatanti-human IgG, Fc γ fragment specific capture antibody. After coatingthe wells were washed twice with PBS/0.05% Tween20 (PBST) and thenblocked for 1 h at RT with PBST containing 5% milk powder followed bytwo washing steps with PBST. Recombinant BCMA and TACI Fc-fusionproteins are added and captured for 1 h at RT. Then the purified anddiluted BAFFR specific Fabs are added and the plates are incubated for 1h at RT. To detect the Fabs, an alkaline phosphatase (AP)-conjugatedgoat anti-human IgG, Fab specific is added and the plates are incubatedfor 1 h at RT. Following each incubation step the wells are washed fivetimes with PBST. For the detection of the AP-conjugates, AttoPhos(Roche) is used according to the manufacturer's instructions.Fluorescence is measured using a TECAN Spectrafluor plate reader.

3. Cell-Based functional assays

BLyS Induced Co-Stimulation of Human B-Cell Proliferation

Untouched B-lymphocytes were purified from peripheral blood mononuclearcells by depletion of other cell types using the MACS B-Cell isolationkit II (Miltenyi Biotec). To induce B-cell proliferation, 100 μlcontaining 1×10⁵ cells were seeded into round-bottom 96well plates infive replicates. Human soluble BLyS was added at a concentration of 3ng/ml together with 0.35% (vol/vol) beads coupled with anti-human IgMantibodies. To determine their inhibitory potencies, anti-BAFFRantibodies at different concentrations were added at differentconcentrations. For the determination of agonistic properties ofanti-BAFFR antibodies, the cells were induced with 0.35% (vol/vol) beadscoupled with anti-human IgM antibodies and increasing concentrations ofanti-BAFFR antibodies (no additional BLyS added). Afterwards, the cellswere cultured for 3 days. For the last 12 hours, 1 μCi/well of tritiatedthymidine was added. Cells were harvested onto a filter and the cellassociated radioactivity was quantified in a scintillation counter.

BLyS Induced Co-Stimulation of Human B-Cell IgG1 Production

Untouched B-lymphocytes were purified from peripheral blood mononuclearcells by depletion of other cell types using the MACS B-Cell isolationkit II (Miltenyi Biotec). To induce IgG1 synthesis, 100 μl containing1×10⁵ cells were seeded into round-bottom 96well plates in fivereplicates and stimulated with 3 ng/ml human soluble BLyS together with100 ng/mL human IL-21 (Pepro Tech Inc.). To determine their inhibitorypotencies, anti-BAFFR antibodies were added at different concentrations.For the determination of agonistic properties of anti-BAFFR antibodies,the cells were induced with 100 ng/mL human IL-21 and increasingconcentrations of anti-BAFFR antibodies (no additional BLyS added). Thecells were cultured for 9 days and the supernatants were collected. IgG1was determined in the cell supernatants by enzyme linked immuno-sorbentassay (ELISA).

Antibody Dependent Cellular Cytotoxicity Assay (ADCC)

Untouched B-lymphocytes were purified from peripheral blood mononuclearcells (PBMC) by depletion of other cell types using the MACS B-Cellisolation kit II (Miltenyi Biotec). Similarly, autologous natural killer(NK) cells were purified from PBMC by negative depletion on an AutoMACsdevice using the MACS human NK Cell Isolation Kit (Milteny Biotech).Increasing concentrations of anti-BAFFR antibodies in 100 μl wereincubated with 1×10⁴ B-cells in 50 μl of culture medium in V-shaped96-well plates (Corning) for 20 minutes. Then, 50 μl containing 1×10⁵ NKcells were added and incubated for 4 hours at 37° C. The cells were spundown and 150 μl of supernatant was removed. The cells were re-suspendedand 10 μl of a 1:100 dilution of 7-amino actinomycin D (7-AAD, BectonDickinson) was added. The cells were enumerated in a FACScalibur (BectonDickinson) instrument. Data analysis of 7-AAD positive cells wasperformed using the CellQuest Pro software (Becton Dickinson).

4. In Vivo Functional Assays

In Vivo B Cell Depleting Assay

Depleting effects of anti-BAFFR antibodies on B cell numbers areassessed as follows:

-   -   a) Relative numbers of B cells in the blood lymphocyte        compartment are measured by staining of whole blood or isolated        peripheral blood mononuclear cells (PBMC) with fluorescent        labelled antibodies specific for B cell and T cell surface        markers (CD3 and CD19, respectively). The relative percentage of        CD3 and CD19 positive cells is quantified by flow cytometry and        a selective reduction of B cells can be expressed by an increase        in the ratio of T cells to B cells.

b) Asolute B cell numbers are determined as cells/microliter of blood byflow cytometry using fluorescent labelled anti-CD19 antibody incombination with TruCOUNT tubes (Cat #340334; Becton Dickinson, San JoseCalif.) according to the manufacturer's instructions.

CIA Animal Model Assay

Collagen induced arthritis (CIA) has been proposed to reflect manyaspects of the human disease. CIA is induced by immunisation ofgenetically susceptible strains of mice with type II collagen inadjuvant and is mediated by autoimmune reactions including autoantibodygeneration, which then bind to a particular region of type II collagen(CII). Both B- and T-lymphocytes are important in the pathogenesis ofCIA. The joint histology in the animal model has many similarities tothe human disease with aspects such as synovial hyperplasia, marginalerosion and destruction of the cartilage surface being some of thefeatures of the pathophysiology.

After immunization with collagen type II (C-II) at day 0 and subsequentbooster treatment with C-II at day 21, DBA11 mice usually develop severesymptoms of arthritis. In the present study, the mice are treatedintraperitoneally, twice weekly with 200 ug per mouse of anti BAFFRantibodies or isotype control antibodies in a prophylactic regimen (daysminus 10 to approximately day 36). Paw swelling is monitored throughoutthe experiment as an indicator of disease severity. Histology isperformed on the paws at the end of the experiment if swelling efficacyis observed. B cell depletion in spleen, blood and lymph nodes can beassessed by cell isolation and FACS anaylsis. ELISA for anti collagenantibodies are performed to investigate whether the antibody titre alsois suppressed. Antibodies are presumed to have good efficacy in a CIAanimal model if they exhibit a statistically significant reduction inswelling when a Dunnetts multiple comparison test (ONE WAY ANOVA) hasbeen applied. Usually this translates to a 50% reduction in paw swellingover the time course with low variation between animals within eachgroup.

EXAMPLES

The following table describes SEQ ID numbers for the corresponding CDRsof specific examples of antibodies of the invention. HCDR1, HCDR2 andHCDR3 stands for the CDR1, CDR2 and CDR3 of the heavy chain of anantibody and LCDR1, LCDR2 and LCDR3 stands for the CDR1, CDR2 and CDR3of the light chain of an antibody.

TABLE 1 Correspondence mAb# and SEQ IDs mAb # HCDR1 HCDR2 HCDR3 LCDR1LCDR2 LCDR3 MOR06743 NO: 2 NO: 9 NO: 16 NO: 23 NO: 30 NO: 37 MOR06654NO: 3 NO: 10 NO: 17 NO: 24 NO: 31 NO: 38 MOR07342 NO: 4 NO: 11 NO: 18NO: 25 NO: 32 NO: 39 MOR07347 NO: 5 NO: 12 NO: 19 NO: 26 NO: 33 NO: 40MOR07348 NO: 6 NO: 13 NO: 20 NO: 27 NO: 34 NO: 41 MOR07349 NO: 7 NO: 14NO: 21 NO: 28 NO: 35 NO: 42

Detailed Characterization of the Examples

Determination of the EC50 of the Antibodies of the Invention in a HumanFab Format in FACS

The EC₅₀ of the Fabs is determined by Fab titration in FACS ontransfected HEK293 cells as well as on Raji cells expressing endogenousBAFFR.

TABLE 2 EC₅₀ of Fab antibodies on Raji cells and transfectedHEK293-hBAFFR EC₅₀ [nM] on EC₅₀ [nM] on MOR0 Raji cells HEK293-hBAFFR6654 0.12/0.32/0.44/0.51 0.98/0.25 6743 0.23/0.18 0.59/0.27 7342 0.060.18 7347 0.03/0.06/0.18 0.08 7348 0.12 0.24 7349 0.13 0.24

Since the Glutamine found in MOR07347 at position 110 in HCDR3 seems tobe advantageous, this position is also introduced in the cross-cloneMOR6743 resulting in the antibody MOR07685. This antibody shows an EC₅₀value determined in FACS titration that is comparable to those ofMOR06743 and MOR07347.

Determination of IC₅₀ values of the Fabs in BAFFR-BLyS binding assay(FACS)

The IC50 values of the antibodies of the invention in a human Fab formatare determined in a BAFFR-BLyS binding inhibition assay by Fab titrationon Raji cells (FACS). The results from these analyses are listed inTable 3.

TABLE 3 IC₅₀ values for BAFFR-BLyS binding inhibition in a human Fabformat on Raji cells MOR0 IC₅₀ [nM] 6654 0.09/0.37/0.30/0.16 67430.13/0.17 7342 0.09 7347 0.02 7348 0.20 7349 0.17 7685 0.12

Determination of IC₅₀ Values of the Antibodies in BAFFR-BLyS BindingAssays (ELISA)

The IC50 values of the antibodies of the invention in a human Fab andIgG2 format are determined in a competitive ELISA in which theantibodies are titrated to inhibit the interaction between theextracellular domain of BAFFR (BAFFR:Fc fusion protein) and humansoluble BLyS. IC₅₀ values were also determined in a similar competitiveELISA in which the antibodies of the invention were titrated to inhibitthe binding of a BAFFR derived peptide (miniBR3) to human soluble BLySassay.

The results from these analyses are listed in Table 4.

TABLE 4 IC₅₀ values for BAFFR-BLyS binding inhibition in a human IgG2format BLys/BAFFR:Fc BLys/BAFFR competition peptide competition ELISAIC₅₀ [nM] ELISA IC₅₀ [nM] MOR0 Hu Fab Hu IgG2 Hu IgG2 6654 1.1  0.0245.9 6743 0.1  0.034 3.5 7342 0.14 n.d. n.d. 7347 0.1  0.019 n.d. 73480.96 n.d. n.d. 7349 0.44 0.05  n.d.

Analysis of Cross-Reactivity to BCMA and TACI

BCMA and TACI are proteins that are related to BAFFR and can also bindthe ligand BLyS. The Fabs are tested for unwanted cross-reactivity tothese two proteins. The cross-reactivity of the binders to BCMA istested on recombinant protein in ELISA and on cell surface antigen inFACS analysis.

In ELISA the Fabs are titrated from 400 nM down to 0.005 nM on BAFFR:Fccaptured to a Maxisorp plate via an anti-human Fc antibody. OnlyMOR07342 and MOR07346 show some cross-reactivity to BCMA at high Fabconcentrations >100 nM. In contrast MOR06654, MOR07347, MOR07348,MOR07349 show no binding to BCMA above background. A 1000-folddiscrimination factor is met by all Fabs.

In FACS the following Fabs are analyzed on BCMA-transfected HEK293 cellsand titrated from 1 μM down to 0.005 nM: MOR06654, MOR06743, MOR07342,MOR07347, MOR7348 and MOR07349. At 330 nM only MOR07342 show an elevatedbinding signal which is 2-fold over background. All other tested Fabsshow no signal at this concentration. At a Fab concentration of 1 μMMOR06654, MOR06743 and MOR07347 showed signals 3-4 fold over background.MOR07342 shows binding to BCMA-transfected cells with a signal 20-foldover background. MOR007348 and MOR07349 do not bind at all to BCMA up toa Fab concentration of 1 μM.

Potency of Antibodies of the Invention in Functional B Cell Assays

Potency and Agonism of Antibodies in the BLyS Mediated Co-StimulatoryB-Cell Proliferation Assay

Primary human blood derived B-cells are stimulated with anti-IgMantibodies and human soluble BLyS to induced B-cell proliferation.Antibodies of the invention are titrated to block the co-stimulatoryeffect of BLyS. In order to measure the agonistic (i.e. BLyS-like)effects of the antibodies, B-cells are stimulated with anti-IgMantibodies alone. BAFFR antibodies are titrated to measure a potentialenhancement of B-cell proliferation. The results from these analyses areshown in Table 5 for different BAFFR antibody formats (human Fab, IgG2and IgG1).

TABLE 5 antagonistic and agonistic activity of antibodies of theinvention in a Fab, IgG2 and IgG1 format B-cell proliferation inhibitionAgonism IC₅₀ EC₅₀ format MOR0 [nM] [nM] Fab 6654 16 >187 Fab 6743 2 >187Fab 7342 0.9 >187 Fab 7347 1.3 >187 Fab 7348 2.9 >187 Fab 7349 3.5 >187IgG2 6654 0.084 >187 IgG2 6743 0.032 >187 IgG2 7342 0.041 >187 IgG2 73470.043 >187 IgG2 7348 0.079 >187 IgG2 7349 0.072 >187 IgG1 66540.122 >187 IgG1 7342 0.031 >187 IgG1 7347 0.034 >187 IgG1 7349 0.079>187

Potency and Agonism of Antibodies in the BLyS Mediated Co-StimulatoryB-Cell IgG1 Production Assay

Primary human blood derived B-cells are stimulated with IL-21 and humansoluble BLyS to induce IgG1 production. Antibodies of the invention aretitrated to block the co-stimulatory effect of BLyS. In order to measurethe agonistic (i.e. BLyS-like) effects of the antibodies, B-cells arestimulated with IL-21 alone. Anti-BAFFR antibodies are titrated tomeasure a potential enhancement of IgG1 secretion. The results fromthese analyses are shown in Table 6.

TABLE 6 IC₅₀ of B-cell IgG1 production B-cell IgG1 production InhibitionIC₅₀ Agonism EC₅₀ format MOR0 [nM] [nM] IgG2 6654 0.028 >62.5 IgG2 67430.018 >62.5 IgG2 7342 0.023 >62.5 IgG2 7347 0.024 >62.5 IgG2 73480.088 >62.5 IgG2 7349 0.039 >62.5

Potency of Antibodies to Elicit B-Cell Killing in the ADCC Assay

Anti-BAFFR antibodies in increasing concentrations are allowed to bindto primary human blood derived B-cells before autologous natural killer(NK) cells are added to induce the killing reaction. After four hours,the number of apoptotic cells is enumerated by FACS. The results fromthese analyses are shown in Table 7 for IgG1 and IgG2 antibody formats.

TABLE 7 EC₅₀ showing B-cell killing activity of antibodies of theinvention in IgG1 and IgG2 format ADCC format MOR0 EC50 [nM] IgG26654 >63 IgG2 7342 >63 IgG1 6654 0.186 IgG1 7342 0.195

Example 2 In Vivo Efficacy in a CIA Mouse Model

Mice were treated with 200 ug/animal of anti BAFFR antibody (conjugatedto murine IgG2a) or isotype control antibody 9, 6 and 2 days beforeimmunization with bovine type 2 collagen in complete Freunds adjuvant.Antibodies were applied at 200 ug/mouse twice a week, throughout theexperiment (until day 35 after immunization). Mice were boosted withbovine type 2 collagen in phosphate buffered saline, 21 days afterimmunization. Swelling was assessed every 2 to 3 days from day of boostonwards. As shown in FIG. 1, anti BAFFR antibody significantly reducedswelling when compared to its control anti CSA antibody.

Example 3 Depletion of Peripheral B Cells in Cynomolgus Monkey

Cynomolgus monkeys were treated with 4 weekly doses of 20 mg/kg i.v. ofthe anti-human BAFF-R monoclonal antibody MOR06654 (IgG1/k). B cellsnumbers in blood were determined at different pre-dose and post dosetime points by FACS analysis. In brief, blood samples were incubatedwith fluorescence-labelled anti-CD20 antibody (Anti-human CD20-PE, Clone2H7, BD, cat #555623) or anti-CD40 antibody (Anti-human CD40-APC, Clone5C3 BD, cat #555591) in True Count tubes (BD, cat #340334). The resultsare shown in FIG. 2.

B cells got rapidly depleted in treated monkeys down to a mean of 33% ofthe pre-dose B cell number (n=3). Reduction of B cells remained orincreased with subsequent doses. The mean of B cell reduction was 85%(n=3) on day 56, i.e., 45 days after the last dose. A gradual increaseof B cell numbers was observed after day 56.

In conclusion, treatment with the human anti-BAFF-R antibody leads to arapid and sustained reduction in peripheral B cells in cynomolgusmonkey. This pharmacodynamic effect is reversible, allow for therestoration of the normal B cell homeostasis after elimination of theantibody from circulation.

Example 4 Stronger and More Sustained B Cell Depletion withNon-Fucosylated Antibodies

In a second experiment, cynomolgus monkeys were treated with a singledose of MOR06654 (IgG1) or the non-fucosylated variant MOR06654B.MOR06654B was produced using the Potelligent™ cell lines (Biowa, Inc.).These cell lines are CHO mammalian cell lines, knocked-out for the geneencoding fucosyltransferase. The antibodies (MOR06654B) produced in suchcell lines are not fucosylated. This time, the single dose was only 20μg/kg i.v.

As shown in FIG. 3, a marginal, but observable reduction of B cells bytreatment with 20 μg/kg i.v. MOR06654 was observed on day 7 (meanreduction 22%, n=3). The decrease in peripheral B cell was morepronounced and more sustained for the antibody MOR06654B, lackingfucose. Here the reduction reached 57% (n=3) and is still about 40%(n=3) 28 days after the single dose.

Example 5 Screening Antibodies that Cross-Block BAFFR Binding Antibodiesof the Present Invention

Biacore Cross-Blocking Assay

The following generally describes a suitable Biacore assay fordetermining whether an antibody or other binding agent cross-blocks oris capable of cross-blocking antibodies according to the invention. Itwill be appreciated that the assay can be used with any of the BAFFRbinding agents described herein.

The Biacore machine (for example the BIAcore 3000) is operated in linewith the manufacturer's recommendations.

BAFFR may be coupled to e.g. a CM5 Biacore chip by way of routinely usedamine coupling chemistry, e.g. EDC-NHS amine couplilng, to create aBAFFR-coated surface. In order to obtain measurable levels of binding,typically 200-800 resonance units of BAFFR may be coupled to the chip(this amount gives measurable levels of binding and is at the same timereadily saturable by the concentrations of test reagent being used).

An alternative way of attaching BAFFR to the BIAcore chip is by using a“tagged” version of BAFFR, for example N-terminal or C-terminalHis-tagged BAFFR. In this format, an anti-His antibody would be coupledto the Biacore chip and then the His-tagged BAFFR would be passed overthe surface of the chip and captured by the anti-His antibody.

The two antibodies to be assessed for their ability to cross-block eachother are mixed in a stochiometrical amount, e.g. at a one to one molarratio, of binding sites in a suitable buffer to create the test mixture.The buffer used is typically a buffer which is normally used in proteinchemistry, such as e.g. PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄,1.76 mM KH₂PO₄, pH 7.4). When calculating the concentrations on abinding site-basis the molecular weight of an antibody is assumed to bethe total molecular weight of the antibody divided by the number oftarget (i.e. BAFFR) binding sites on that antibody.

The concentration of each antibody in the test mixture should be highenough to ensure saturation of the binding sites for that antibody onthe BAFFR molecules which are bound on the BIAcore chip. The antibodiesin the mixture are at the same molar concentration (on a binding basis)and that concentration would typically be between 1.0 mM and 1.5 mM (ona binding site basis).

Separate solutions containing the separate antibodies on their own arealso prepared. The buffer used for these separate solutions should bethe same buffer and at the same concentration as was used for the testmixture.

The test mixture is passed over the BAFFR-coated BIAcore chip and thebinding recorded. The bound antibodies are thereafter removed bytreating the chip with e.g. an acid, such as 30 mM HCl for about 1minute. It is important that the BAFFR molecules which are bound to thechip are not damaged.

The solution of the first antibody alone is then passed over theBAFFR-coated surface and the binding is recorded. Thereafter, the chipis treated to remove all of the bound antibody without damaging thechip-bound BAFFR, e.g. by way of above mentioned acid treatment.

The solution of the second antibody alone is then passed over theBAFFR-coated surface and the amount of binding recorded.

The maximal theoretical binding can be defined as the sum of the bindingto BAFFR of each antibody separately. This is then compared to theactual binding of the mixture of antibodies measured. If the actualbinding is lower than that of the theoretical binding, the twoantibodies are cross-blocking each other.

Elisa-Based Cross-Blocking Assay

Cross-blocking of an anti-BAFFR antibody or another BAFFR binding agentmay also be detected by using an ELISA assay.

The general principle of the ELISA-assay involves coating an anti-BAFFRantibody onto the wells of an ELISA plate. An excess amount of a second,potentially cross-blocking, anti-BAFFR antibody is then added insolution (i.e. not bound to the ELISA plate). A limited amount ofBAFFR-Fc is then added to the wells.

The antibody which is coated onto the wells and the antibody in solutionwill compete for binding of the limited number of BAFFR molecules. Theplate is then washed to remove BAFFR-Fc that has not bound to the coatedantibody and to also remove the second, solution phase, antibody as wellas any complexes formed between the second, solution phase antibody andBAFFR-Fc. The amount of bound BAFFR is then measured using anappropriate BAFFR detection reagent. An antibody in solution that isable to cross-block the coated antibody will be able to cause a decreasein the number of BAFFR molecules that the coated antibody can bindrelative to the number of BAFFR molecules that the coated antibody canbind in the absence of the second, solution phase, antibody.

This assay is described in more detail further below for two antibodiestermed Ab-X and Ab-Y. In the instance where Ab-X is chosen to be theimmobilized antibody, it is coated onto the wells of the ELISA plate,after which the plates are blocked with a suitable blocking solution tominimize non-specific binding of reagents that are subsequently added.An excess amount of Ab-Y is then added to the ELISA plate such that themoles of Ab-Y BAFFR binding sites per well are at least 10 fold higherthan the moles of Ab-X BAFFR binding sites that were used, per well,during the coating of the ELISA plate. BAFFR-Fc is then added such thatthe moles of BAFFR-Fc added per well are at least 25-fold lower than themoles of Ab-X BAFFR binding sites that were used for coating each well.Following a suitable incubation period, the ELISA plate is washed and aBAFFR detection reagent is added to measure the amount of BAFFRspecifically bound by the coated anti-BAFFR antibody (in this caseAb-X). The background signal for the assay is defined as the signalobtained in wells with the coated antibody (in this case Ab-X), secondsolution phase antibody (in this case Ab-Y), BAFFR buffer only (i.e. noBAFFR) and BAFFR detection reagents. The positive control signal for theassay is defined as the signal obtained in wells with the coatedantibody (in this case Ab-X), second solution phase antibody buffer only(i.e. no second solution phase antibody), BAFFR and BAFFR detectionreagents. The ELISA assay needs to be run in such a manner so that thepositive control signal is at least 6 times the background signal.

To avoid any artifacts (e.g. significantly different affinities betweenAb-X and Ab-Y for BAFFR) resulting from the choice of which antibody touse as the coating antibody and which to use as the second (competitor)antibody, the cross-blocking assay needs to be run in two formats: 1)format 1 is where Ab-X is the antibody that is coated onto the ELISAplate and Ab-Y is the competitor antibody that is in solution and 2)format 2 is where Ab-Y is the antibody that is coated onto the ELISAplate and Ab-X is the competitor antibody that is in solution.

The invention claimed is:
 1. A method of treating an autoimmune diseasecomprising the step of administering to a subject a compositioncomprising an isolated antibody or antigen-binding fragment thereof,wherein said antibody or antigen-binding fragment thereof comprisesHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the sequences of said CDRsare selected from the group comprising: a) SEQ ID NOs: 2, 9, 16, 23, 30,and 37, respectively; b) SEQ ID NOs: 3, 10, 17, 24, 31, and 38,respectively; c) SEQ ID NOs: 4, 11, 18, 25, 32, and 39, respectively; d)SEQ ID NOs: 5, 12, 19, 26, 33, and 40, respectively; e) SEQ ID NOs: 6,13, 20, 27, 34, and 41, respectively; and f) SEQ ID NOs: 7, 14, 21, 28,35, and 42, respectively; and wherein said antibody or antigen-bindingfragment thereof binds BAFF-R.
 2. The method of claim 1, wherein theautoimmune disease is selected from rheumatoid arthritis, systemic lupuserythematosus, Sjogren's Syndrome, multiple sclerosis and pemphigusvulgaris.
 3. The method of claim 1, wherein the subject is human.
 4. Themethod of claim 1, wherein the antibodies or antigen-binding fragmentsthereof comprise, respectively, a variable heavy chain comprising thesequence of a) SEQ ID NO: 51; b) SEQ ID NO:52; c) SEQ ID NO:53; d) SEQID NO:54: e) SEQ ID NO:55; or f) SEQ ID NO:56.
 5. The method of claim 1,wherein the antibodies or antigen-binding fragments thereof comprise,respectively, a variable light chain comprising the sequence of: a) SEQID NO: 44; b) SEQ ID NO:45; c) SEQ ID NO:46; d) SEQ ID NO47: e) SEQ IDNO:48; or f) SEQ ID NO:49.